Insulin levels are inadequate to maintain euglycemia in patients with type-2 diabetes mellitus (T2DM) and in animal models of the disease, which is due in part to perturbations in ?-cell Ca2+ homeostasis. TALK-1 chan- nels are key regulators of pancreatic ?-cell electrical excitability, Ca2+ handling and glucose-stimulated insulin secretion (GSIS). KCNK16 the gene that codes for TALK-1 is the most abundant K+ channel transcript of the ?-cell and TALK-1 is the most islet-restricted ion channel. Moreover, a mutation in KCNK16 results in neonatal diabetes and a nonsynonymous polymorphism in KCNK16 causes a predisposition for developing T2DM. However, our ability to utilize TALK-1 as a therapeutic target to normalize ?-cell Ca2+ homeostasis in T2DM has not been determined. The long term goal of this research is to determine the therapeutic potential of targeting TALK-1 for treating diabetes. The objective of this project is to identify how TALK-1 influence ?-cell Ca2+ han- dling, insulin secretion and glucose homeostasis. This project will test the central hypothesis that TALK-1 inhi- bition normalizes ?-cell Ca2+ homeostasis under diabetic conditions preventing ?-cell dysfunction and maintain- ing euglycemia. This project is supported by strong preliminary data that has identified TALK-1 an important determinant of human and rodent ?-cell ER Ca2+ handling and insulin secretion. Further data that has opti- mized a thallium (Tl+) based fluorescent assay for a high throughput screen (HTS) of human TALK-1 to identify small-molecule probes of this channel; pitot screens (3248 small molecules) also identified the first pharmacol- gocial probes of TALK-1 activity. The rationale that underlies this project is that understanding how ?-cell func- tion is influenced by TALK-1 channel activity with genetic and small-molecule probes will expose novel thera- peutic targets for treating T2DM. This project will be accomplished with the following two specific aims: 1)De- velop potent and selective small-molecule probes of TALK-1 channels; and 2) Determine the therapeutic po- tential of targeting ?-cell TALK-1 channels for treating diabetes. Under the first aim, small-molecule probes of TALK-1 will be identified with a Tl+ assay based HTS of human TALK-1. This aim will utilize iterative parallel synthesis and verification approaches, to optimize TALK-1 small-molecule probes; testing selectivity (with TALK-1 knockout mouse ?-cells), potency, toxicity and drug metabolism pharmacokinetic (DMPK) profiles with assays such as Tl+ flux, Ca2+ flux, cytotoxicity and tier one in vitro DMPK. Under the second aim, conditionally ablating, blocking or pharmacologically modulating TALK-1 channels in mouse and human ?-cells will be uti- lized to determine how these channels influence ER Ca2+ stores, cytoplasmic Ca2+ homeostasis, membrane potential, GSIS, and glucose homeostasis. The roles of ?-cell TALK-1 will be assessed under physiological conditions as well as under the stressful conditions associated with diabetes both in vivo and in vitro. This pro- ject is significant because it is expected to lead to pharmacological strategies for treating diabetes; it is essen- tial for uncovering novel ?-cell selective therapies for normalizing ER Ca2+ homeostasis in diabetic conditions.